Nonisotopic colorimetric and chemiluminescent technologies will be developed for use with ribonuclease protection assays. The ribonuclease protection assay is an increasingly utilized technique in molecular biology affording sensitive and quantitative measurement of mRNA levels. The ability of the assay to detect multiple mRNA levels simultaneously makes this technique a unique and powerful tool for use in research and diagnostic applications. In a ribonuclease protection assay, autoradiography of the polyacrylamide gel-resolved products represents the endpoint of the experiment. However, all of the available nonisotopic detection protocols require blotting of nucleic acids onto a sold support prior to detection. Therefor, we propose an approach which will circumvent this requirement and allow detection directly in the gel used to resolve the nucleic acids. Our initial studies demonstrated that in vitro transcripts containing 4-thiouridine are both functional in ribonuclease protection assays and can be detected in gel down to 1 picomole by a colormetric detection protocol using N- ethylamaleimide and base. During phase I we will synthesize a derivative of N-ethylmaleimide which has been reported to greatly potentiate the sensitivity of thiol detection. Furthermore, in an effort to extend the sensitivity of ribonuclease protection assays beyond that attainable with radioactivity, we will synthesize a thiolated derivative of the chemiluminescent molecule isoluminol. This derivative will be coupled to either 4- thiouridine- or 5 mercuriuridine-substituted transcripts and detected using a non-enzymatic, but catalytic, oxidation system with molecules which can all readily diffuse in and out of a polyacrylamide gel matrix. Once we have developed a nonisotopic ribonuclease protection assay, it will be utilized with a panel of oncogene and tumor suppressor gene probes which are used extensively at Ambion for the development of single base mismatch technologies. Ultimately, the technologies which we develop will have wide applications in molecular biology research extending beyond ribonuclease protection assays.